The use of virus-based vectors has become a crucial delivery method for both in vitro applications in drug discovery, in vivo and ex vivo clinical assays and for gene therapy. Viral vectors fall into two main categories: integrating vectors, which insert themselves into the recipient genome and non-integrating vectors, which usually form an extra chromosomal genetic element. Integrating vectors such as gamma-retroviral vectors (RV) and lentiviral vectors (LV) are stably inherited. Non-integrating vectors, such as adenoviral vectors (ADV) and adeno-associated virus (AAV) vectors are quickly lost from cells that divide rapidly. Some factors influencing the choice of a particular vector, include its packaging capacity, its host range, its gene expression profile, its transduction efficiency and its capacity to elicit immune responses, which is particularly problematic if repeated administrations or transductions are needed. Some of these parameters can be adjusted or controlled. One parameter is the use of highly concentrated but also highly purified vectors to allow efficient cell transduction and to avoid specific cell responses due to contents other than the vector itself.
Current methods used to produce and concentrate the virus based vectors are not optimal to preserve the vector integrity and the batch quality. Indeed, small-scale experimental batches are commonly concentrated by simple methods based on ultracentrifugation or centrifugation on ready-to-use central units. Such batches are referred to herein as batches A-Serum (A-S) and B-Serum (B-S) and the processes used to produce such batches is described in FIG. 4A. Those methods also concentrate cellular debris, membrane fragments and proteins secreted by the producer cells and from the culture medium including serum and are unsuitable for producing vector batches under good manufacturing practices (GMP). One major drawback of these batches is their inability to allow high transduction efficiency in a reproducible manner of some non-proliferating cells such as neurons, macrophages or hematopoietic stem cells when using low or medium multiplicity of infection (M.O.I.). Usually, scientists focused on vector pseudotyping or transduction protocol optimizations to improve the transduction efficiency (Janssens et al., 2003) although the use of higher M.O.I. is the clue to reaching high transduction levels. However, since such a batch B-S induces cell toxicity (Selvaggi et al., 1997; Reiser, 2000; Baekelandt et al., 2003), the results of transduction efficiency with this type of product B-S are always a balance between the transduction level and the resulting toxicity on target cells. Furthermore, another drawback of published retroviral or lentiviral vectors concentrated by classical techniques is the inability of transduced stem cells, particularly for hematopoietic stem cells, to progress down differentiation pathways after transduction.
Merten et al. (2010) used a downstream process based on several membrane-based and chromatographic steps but with a production process using a medium with 10% of serum, which is a critical difference between the process of Merten et al. and the process developed according to the present invention. The present invention provides compositions and methods for transduction of cells using retroviral or lentiviral vectors which exhibit a high purity level. Such compositions and methods have no detrimental impact on stem cell differentiation into specialized cells.
The production step has a great impact on the final concentrated product as it provides the starting material to be subsequently subjected to concentration and purification steps. Production might be performed with or without serum, with or without sodium butyrate induction and the supernatant can be harvested either once 48 h after transfection or twice 64 h and 88 h post transfection for example (Cooper et al., 2011). The major disadvantage of such harvesting times is the lack of consideration of the vector particle half life. These conditions have a great impact on the content of initial contaminants (DNA and/or protein contaminants) and the level of toxicity content of the crude supernatant. These elements must be measured to characterize each batch corresponding to a specific process of production, purification and concentration i.e batches A, B, C and D of the present invention. Cooper et al. characterized neither the initial product nor the purified final product by measuring initial contaminants and their removal after concentration/purification process, contrary to the present invention (See Table 1)
The present invention provides a final purified RNA based viral vector composition comprising less than 2% of initial protein contaminants and less than between 70 and 90% of initial DNA contaminants, compared to the crude RNA based viral vector composition as present in the cell serum-free medium, said composition being capable of transducing eukaryotic cells without significantly affecting cell viability.
The present invention provides a purified RNA based viral vector composition comprising less than 2% of initial protein contaminants and less than 30% of initial DNA contaminants, compared to the crude RNA based viral vector composition as present in the cell serum-free medium, said composition being capable of transducing eukaryotic cells without affecting cell viability.
Applicants demonstrate herein that each of these parameters (serum, sodium butyrate induction and vector harvest times) modify the initial crude vector supernatant composition which induces a differential toxicity level on target cells.